Preparation and application of transhintotalphenolic acid

ABSTRACT

A process for preparing tanshintotalphenolic acid and the use of the product are disclosed. The process comprises: tanshin is hot-extracted with water, the extract is separated and refined by polyamide column and macroporous adsorption resin column, and lyophilized to obtain tanshintotalphenolic acid. The yield of the final end product is more than 4 percent based on the amount of crude drug and the content of totalphenolic acid is more than 80 percent. The tanshintotalphenolic acid obtained can be used as the medicine for preventing and treating cerebrovascular and cardiovascular diseases and so on.

FIELD OF THE INVENTION

The present invention relates to an extract of Traditional ChineseMedicine (TCM). More particularly, the present invention relates to arefined total phenolic acid extracted from TCM Danshen and the methodfor extracting the same.

BACKGROUND OF THE INVENTION

Danshen, also known by its botanical name Radix Salvia Miltiorrhizae, isone of the most common-used traditional Chinese medicines (TCM) inChina. It mainly consists of two categories of chemical ingredients,namely the water-soluble and the non water-soluble. Dating back to theearly 20^(th) century, the chief research on these ingredients hasalways been concentrated on the non water-soluble ones represented bytashinone, and achieved great success after decades of efforts. It isnot until the beginning of 1980s that, after a lot of work, ourscientists have studied Danshen's water-soluble ingredients, and firstreported structure of the water-soluble one, Danshensu. Afterward, tensof water-soluble ones have also been discovered one after another withthe definitive chemical structure. Subsequently it has been proven that,of the active ingredients in Danshen's water-soluble ones, the mosteffective is phenolic acid compounds, such as salvianoic acid A(1),B(2), C(3), D(4), E(5), F(8), G(9), H(11), I(12), J(13), rosmarinci acid(6), alkannic acid (7), isosalvianoic acid C(10), glucoside ofrosmarinci acid(14), etc. (Lian-Niang Li, J. Chinese PharmaceuticalSciences 1997, 6, 57-64). Pharmacologically, a variety of activities ofthese salvianolic acid compounds have already been reported. Forexample, Salvianolic acid A has significant protecting effects oncardiac muscle cell caused by ischemic reperfusion (I/R), andtotalphenolic acid has strong anti-arrhythmia effect induced by I/R;Salvianolic acid A, B and totalphenolic acid have showed markedlyprotective effects against brain damage caused by I/R in rats bylowering content of MDA in brain tissues; there are plenty more othereffects for the salvianolic acid as follows: anti-thrombus effects,protective effect on liver and kidney, anti-oxidation and inhibitinglipid super oxidation, as well scavenging puperoxide anion and freeradical etc. (Du Guanhua et al, Basic medical science and clinics, 2000,20 (5): 10˜14).

At present, a great number of processes of extracting Salviamolic acidhave been reported, but most of them are mainly focused on passing resincolumn following extracting by water. For example, Takashi Tanaka et alrevealed the method of extracting salvainolate (Chemical PharmaceuticalBulletin, 1989, 37 (2), 340˜344). Besides, there have also been manyother scientists who have adopted similar methods for extractingphenolic compounds from Danshen, such as Koji Hase et al (Planta Medica,1997, 63, 22˜26), Xu Yaming et al (China patent CN1247855A, published inMarch, 2000), Liu Ping et al (China patent CN1270809A, published inOctober, 2000), and Li Lianniang (China Patent Application No.0114228.2, filed on September 2001). But all of above-mentionedprocesses for extracting have a common problem in industrialization,namely a great deal of water need to be concentrated. Because of theinstability, the concentrating temperature of total salvainolic acidwater decoction must be varying between 50° C. and 60° C., whichaccordingly will result in both the difficulties in techniques and risein cost. Meanwhile, the lasting heating process, although between 50° C.and 60° C., will also produce a series of serious problems includinginstability, and therefore affect its quality and curative effect.Finally, all these problems make it almost impossible for theindustrialization. Another shortcoming of these already-existingprocesses is that the low yield, generally between 2% and 3%, limits itsapplication in the industry.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is, obviating thedrawbacks of the prior art, to provide a method for preparation oftotalphenolic acid with high yield, low cost, good quality, andconvenience of being applied in the industry.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention of preparing totalphenolic acid mainly consists ofthe following steps, namely decocting Danshen with hot water; separatingthe decoction with Polyamide column and macroporous adsorption resin.

The totalphenolic acid may be obtained by the following scheme:

-   (a) After the impurities is eliminated, the Danshen is cut into    little sections or pulverized into crude powder, and decocted with    hot water. The decoction is filtered after its pH value being    adjusted to acidity.-   (b) Applying said decoction on a polyamide column, and washing the    column with water to neutral condition. Eluting the column with weak    basic aqueous solution, and collecting the eluent.-   (c) Applying the eluent on the macroporous adsorption resin column,    after acidifying the basic eluent of step (b). At first, washing the    column with water to neutral condition, and then eluting the column    with hydrous or anhydrous lower alcohol. Afterward, collecting the    eluent.-   (d) Concentrating the eluent under reduced pressure until there is    no ethanol, and drying it to obtain the total salvianolic acid.

In step (a), Danshen is extracted with hot water for 2 to 4 times, 0.5to 2 h each, and the extracting temperature is 60 to 100° C., preferably90 to 100° C.; the extract solution after each extracting process, aloneor combined, preferably combined, is further treated; the pH value isadjusted with acid to less than 4, preferably below 2.

In step (b), the concentration of weak basic aqueous solution preferablyis from 0.01% to 2%, most preferably 0.08% to 0.5%, and the commonpolyamide materials is used, for example, polycaprolactam (nylon-6).

In step (c), the common macroporous adsorbent resin is used, for examplestyrene-type adsorbent resin; the pH value of weak basic fractions areadjusted by acid to below 4, preferably below 2; the number of carbonatoms in lower ethanol ranges from 1 to 5, for example, the methanol,ethanol, etc.; the eluting concentration is 40% to 95%, preferably 60%to 95%. However, in view of the safety in large-scale industrializedproduction, lowering cost and process simplification, eluting by 95%ethanol is the best option to achieve satisfying purpose.

In step (d), if necessary, said concentrate is dried, such as bylyophilization, following being filtered with microporous filtermembrane.

The totalphenolic acid produced by the method of this invention can beformulated into any kind of pharmaceutically acceptable dosage form, andcan also be combined with other medicaments or active ingredients.

According to this invention, a totalphenolic acid of Danshen, producedby the method of this invention.

A pharmaceutical composition, including the totalphenolic acid ofDanshen of this invention and pharmaceutically acceptable carrier orexcipient.

Compared with the prior art, this invention has the followingadvantages:

1. Easiness of industrialization. In the prior art, a great deal ofwater need to be concentrated during the process, resulting in thedifficulties in industrialization. Such a defect is overcome in thepresent invention, wherein, without heating and decompression, lot ofwater are surprisingly removed. Consequently, the processes andconditions are optimized with no energy consuming and environmentpollution. So, with the advantages in respect of techniques orenvironmental protection, it is easy to bring about industrialization.

2. Reduction of loss in active ingredients. The present invention caneffectively avoid a loss of active ingredients caused by precipitationwith alcohol in prior art, and also prevent instability of totalphenolicacid in concentrating a lot of water. All these above would in effectavoid the losses and decomposition of the active ingredients during theprocess, so as to assure the stability of final product.

3. High yield. The yield of the products produced in the prior art (i.e.the dried totalphenolic acid powder) is only 2% ˜3% by weight based onthe crude herbal medicine; while using the method of present invention,the yield is more than 4%, apparently better than that of the prior art.Moreover, the content of totalphenolic acid in the high quality finalproduct is more than 80% with less impurity.

4. Lower cost. In the method of present invention, a great deal of watercan be concentrated and removed without heating and decompression,effectively reducing the energy consumption and cost. In addition, theyield of the total phonelic acid by this invention is higher, andmoreover its content in the final product is close to or higher thanthat of the prior art, that is to say, more products with equal orbetter quality will be produced from the same amount of crude herbalmedicine. The lower cost will benefit a lot, not only in theindustrialization, but also the patients' economic interests,accordingly bringing about a great social benefit.

5. The studies on animals also show the good effects of thetotalphenolic acid produced by the method of present invention.

Protective Effect of the Totalphenolic Acid Against Cerebral ArteryIschemia in Rats

1. Materials and Methods

1) Animals: Male Wister rats, weighing from 200 to 220 g (CertificateNo. SCXK(Beijing) 2002-003), are obtained from the Animal Center ofBeijing Medical University.

2) Reagents: Chloral Hydrate purchased from Shenyang reagent factory,Liaoning, China, the batch number being 920401.

-   -   Red tetrazoline (TTC) from Beijing chemical plant, the batch        number being 810911.

3) Apparatus: High-frequency electric knife purchased from Beijingmedical electronic apparatus factory.

-   -   The SXP-1B operating microscope from Shanghai medical optical        instrument factory.

4) Tested agents: The total phonetic acid produced by Tianjin TaslyModern TCM institute according to the method of the present invention;and

-   -   The total salvianolic acid produced by Institute of Materia        Medica, Chinese Academy of Medical Sciences in accordance with        the method of China Patent Application No. 01142288.2 filed on        September, 2001.    -   Xiangdan injection, as control drug, purchased from Ya an Sanjiu        pharmaceutical Co. ltd, the bath number being 010901.

5) Formulation method of the tested agents and route of administration:The totalphenolic acid and Xiangdan injection are diluted into desiredconcentration with sterilized physiological saline, 10 mg/kg and 20mg/kg for the totalphenolic acid, and 1 ml/kg (equal to 1 g/kg) forXiangdan injection. By sublingual vein, both of these kinds of drugs areadministered 30 minutes after ischemia.

6) Groups: All rats are randomly divided into the following groups: shamoperation control group, ischemic control group, total phonetic acid(Institute of Materia Medica) 10 mg/kg and 20 mg/kg groups, totalphonelic acid (Tasly) 10 mg/kg and 20 mg/kg groups.

2. Method

1) cerebral artery blockade (electric coagulation) ischemia in rats:Rats are anesthetized by intraperitoneal injection of Trichloraceticaldehyde, 350 mg/kg body weight, and fixed on a board in a left lateralposition. Under the operating microscope, the skin is incised open viamidline between the external auditory meatus and the canthus. Thezygomatic orthopedics is exposed, and removed thoroughly withorthopedics rongeur. The fascia is nipped off along skull, and temporafossa is exposed. Between the squamous orthopedics and mandible isgently propped up with retractor, and at the bottom of the skull theskull window is opened so as to uncover cerebral middle artery. Themiddle artery is burnt out with the high-frequency electric knife inorder to block the blood flow, forming a model of cerebral localischemia. After 30 minutes, the rats are administrated throughsublingual vein, and sent back to cage for feed. The room temperature isrigorously kept between 24° C. and 25° C.

2) The measurement of cerebral infarct volume: 24 hours after thecerebral middle artery is blocked, the rats are beheaded and theircerebrums are taken out. The whole cerebrum is kept at 4° C. in a beakerfilled with normal-saline in a refrigerator for 10 minutes, and then theolfactory bulb, the cerebellum and the low-set brain stem are removed.Along the coronal plane, the cerebrum is chipped into 5 slices, and putinto 5 ml dyeing solutions containing 1.5 ml of 4% TTC and 0.1 ml of 1mol/L di-potassium hydrogen phosphate, light-proof incubated in waterbath 37° C. for 30 minutes. The slices of cerebrum are taken out and putinto 10% formalin for solidification. As a result, the normal cerebraltissue is rose pink, and the ischemic one is white. The weightplanimetry is used to measure the area of infarct, and further thepercentage of the area of infarct to the whole cerebrum hemisphere iscalculated.

3) The measurement of content of water in the cerebrum: 24 hours afterblockade of the cerebral middle artery, the rats are executed, and thewhole cerebrum is gently taken out. After that the olfactory bulb, thecerebellum and the low-set brain stem are taken away, the cerebrum isweighed (regarded as the cerebral wet weight). After that, the cerebrumis dried in an oven at 105° C. till that the weight is constant (about48 hours), and is weighed again (referred to as the cerebral dryweight). The final content of water in the cerebrum is calculated withthe following formula:the content of water in the cerebrum=(the cerebral wet weight−thecerebral dry weight)/the cerebral wet weight×100%.3. Result

Protective effect of the totalphenolic acid against the cerebral middleartery coagulation ischemia in rats dosage number group mg/kg of animalN Volume of infarct (%) the content of water (%) sham operation controlgroup 12 0  79.4586 ± 0.3402** ischemic control group 10 7.0194 ± 4.389 80.4487 ± 0.8614  Xiangdan injection 1 ml/kg 12  0.7553 ± 2.2188** 79.4364 ± 0.5061** total phonelic acid (Institute of 10 12 3.2919 ±3.205#  79.4723 ± 0.5475** Materia Medica) total phonelic acid(Institute of 20 12  1.5156 ± 2.7602* 79.4806 ± 0.6819* Materia Medica)total phonelic acid (Tasly) 10 10  2.8170 ± 3.2621# 79.4529 ± 0.7693*total phonelic acid (Tasly) 20 10  1.5328 ± 3.2575* 79.5914 ± 0.5843*note:(1) **P < 0.01, *P < 0.05, two-sided test, comparing with ischemiccontrol group. #P < 0.05, one-sided test, compared with ischemic controlgroup.(2) 1 ml/kg the Xiang dan injection as a control is equal to 1 g/kg ofcrude herbal medicine;10 mg/kg totalphenolic acid (Tasly) is equal to 0.25 g/kg of crudeherbal medicine (calculated with yield being 4%); and 20 mg/kgtotalphenolic acid (Tasly) is equal to 0.5 g/kg of crude herbal medicine(calculated with yield being 4%).4. Conclusion

By using the method of coagulation in middle cerebral artery to attaincerebral ischemia in rats, the protective effect of the totalphenolicacid produced respectively by Tasly and Institute of Materia Medica oncerebral artery ischemic has been observed. The result revealed that, 30min after administration in vein, 10 mg/kg and 20 mg/kg of both twokinds of phenolic acids could markedly alleviate cerebral edema andcerebral infarction caused by ischemia. Moreover, the said two kinds ofphenolic acids had the same effect administered in the same dosage. Allabove studies have showed that two phenolic acids had the same effect ofanti-cerebral ischemia.

The totalphenolic acid of this invention can be formulated intopharmaceutically acceptable dosage forms, including tablet, capsule,granules, oral liquid, sustained-release formulation, control-releaseformulation, gel, ointment, salve, cream, suppository, injection,powder, patch, dripping pill and suspension.

The totalphenolic acid of this invention can be used for the treatmentof diseases, including cardiovascular and cerebrovascular disease,nephrosis, hepatopathy, pneumonia, pneumocardial disease, pancreatitis,diabetes mellitus, cervical syndrome, ocular fundus vascular disease,ocular fundus neuro-disease, migrain, chronic gastritis, dizziness,orthopedics disease, mountain sickness and senile dementia.

EMBODIMENTS

The following examples are offered for purposes of illustration only andare not intended to limit the scope of the invention in any way.

COMPARATIVE EXAMPLE

Totalphenolic acid is prepared according to the Chinese PatentApplication No. 01142288.2, filed on September 2001.

5 Kg of Danshen herb is ground into crude powder, and is extracted at100° C. for three times with deionized water added, specifically,extracted for 1 hour with 30 L water added the first time, and extractedfor 0.5 hour with 15 L water added the second and third timesrespectively. The extract is concentrated to 5 L at 50° C. under reducedpressure and cooled. Into the concentrate 14 L of 95% ethanol is added.The mixture is allowed to stand over night and filtered. Under reducedpressure, the ethanol is recovered at 50° C. The obtained concentrate isapplied on RA macroporous adsorption resin (mainly containing styreneand chrysophenine, and the weight of dried resin is 2 kg). The resin iswashed with deionized water until that the eluent had no apparentα-naphthol reaction, and then eluted with 50% ethanol until that theeluent had no obvious phenolic hydroxyl reaction with ironsesquichloride potassium ferricyanide added. The fractions areconcentrated at 50° C. under reduced pressure. The mixture is allowed tostand over night in a refrigerator, and filtered to produce the extractof Total salvianolic acid. The pH of the said extract is adjusted with2% sodium hydroxid to 6.5, and the extract is freeze dried to produce114 g of totalphenolic acid. The yield of the final product in crudedrug is 2.3%. The analysis showed that the content of totalphenolic acidin final product amounted to 83.72%, and Salvianolic acid B was inamount of 54.41%.

Total salvianolic acid and Salvianolic acid B are analyzed according tothe method of Chinese Patent Application No. 01142288.3 filed onSeptember 2001.

-   -   (1) Salvianolic acid B: analyzed by HPLC at 288 nm. The        Salvianolic acid B CRS is manufactured by Modern TCM Institute        under Tianjin Tasly Group with purity of 98.0%.    -   (2) Total salvianolic acid: Content=F(A−B)+B        wherein, A is the content of Total salvianolic acid calculated        with the Salvianolic acid B as CRS by ultraviolet        spectrophotometry;    -   B is the content of Salvianolic acid B by HPLC;    -   F is correction factor 0.626.

EXAMPLE 1

5 kg of Danshen herb is ground into crude powder, and is extracted at100° C. at the state of lightly boiling for three times with deionizedwater, specifically, extracted for 1 hour with water (×5.5 fold) addedthe first time, and extracted for 0.5 hour with water (×3 fold) addedthe second and third times respectively. The extract is combined, andthe pH thereof is adjusted with 10% hydrochloric acid to 2.0. Theextract is filtered, and the filtrate is loaded on polyamide column (theamount of the dry resin is two-thirds of that of the crude herb). Thecolumn is washed with deionized water (×5 fold), and the washing isdiscarded. Then the column is eluted with 5 column volumes of the 0.1%aqueous solution of sodium bicarbonate. The fraction was collected,adjusted with 10% hydrochloric acid to pH 2.0, and is loaded on D₁₀₁macro-porous resin column. The column is washed with deionized water toneutral condition and the washing is discarded. Then the column iseluted with 95% ethanol, and the colored belt is collected when it iseluted down. The fractions are concentrated under reduced pressure toentire dryness. The above concentrate is dissolved with water. Themixture is allowed to stand over night in refrigerator, and filtered by0.3 μm mixed cellulose microporous membrane to produce extractionsolution of Total salvianolic acid. Immediately after this totalphenolicacid is adjusted with 2% sodium hydroxide to pH 6.0, it is freeze driedto produce 221 g of freeze-dried powders of Total salvianolic acid. Theyield of the final product is 4.4% based on the amount of the crudeherb. The analysis according to the method of Chinese Patent ApplicationNo. 01142288.2 filed on September 2001 shows that, the totalphenolicacid amounts to 83.94%, and Salvianolic acid B is 53.73% in the finalproduct.

EXAMPLE 2

5 kg of Danshen herb is ground into crude powder, and is extracted at80° C. for three times with deionized water, specifically, extracted for2 hours with water (×5.5 fold) added the first time, and extracted for 1hour with water (×3 fold) added the second and third times respectively.The extracts are combined, and the pH thereof is adjusted with 5%sulfuric acid to 1. The extract is filtered, and the filtrate is loadedon polyamide column (the amount of the dry resin is two-thirds of thatof the crude herb). The column is washed with deionized water (×5 fold),and the washing is discarded. Then the column is eluted with 4 columnvolumes of the 0.2% aqueous solution of sodium bicarbonate. Thefractions are collected, adjusted with 5% sulfuric acid to a pH of 1,and is loaded on AB-8 macro-porous adsorption resin column. The columnis washed with deionized water to neutral condition and the washing isdiscarded. Then the column is eluted with 60% ethanol, and the coloredbelt is collected when it is eluted down. The fractions are concentratedunder the reduced pressure until it had no smell of ethanol. The mixtureis allowed to stand over night in refrigerator, and filtered by 0.3 μmmixed cellulose micro-porous membrane to produce extraction solution ofTotal salvianolic acid. Immediately after this totalphenolic acid isadjusted with 2% sodium hydroxide to pH 6.0, it is freeze dried toproduce 227 g of freeze-dried powders of Total salvianolic acid. Theyield of the final product is 4.5% based on the amount of the crudeherb. The analysis according to the method of Chinese Patent ApplicationNo. 01142288.2 filed on September 2001 shows that, the totalphenolicacid amounts to 83.15%, and Salvianolic acid B is 54.03% in the finalproduct.

EXAMPLE 3

5 kg of Danshen herb is ground into crude powder, and is extracted at100° C. at the state of lightly boiling for three times with deionizedwater, specifically, extracted for 1 hour with water (×5.5 fold) addedthe first time, and extracted for 0.5 hour with water (×3 fold) addedthe second and third times respectively. The extract is combined, andthe pH thereof is adjusted with 10% hydrochloric acid to 2.0. Theextract is filtered, and the filtrate is loaded on polyamide column (theamount of the dry resin is two-thirds of that of the crude herb). Thecolumn is washed with deionized water (×5 fold), and the washing isdiscarded. Then the column is eluted with 5 column volumes of the 0.1%aqueous solution of sodium bicarbonate. The fractions are collected,adjusted with 10% hydrochloric acid to a pH of 2.0, and is loaded on RAmacro-porous resin column. The column is washed with deionized water toneutral condition and the washing is discarded. Then the column iseluted with 60% Methanol, and the colored belt is collected when it iseluted down. The fractions are concentrated under the reduced pressureuntil it had no smell of ethanol. The mixture is allowed to stand overnight in refrigerator, and filter by 0.3 μm mixed cellulose micro-porousmembrane to produce extraction solution of Total salvianolic acid.Immediately after this totalphenolic acid is adjusted with 2% sodiumhydroxide to pH 6.0, it is freeze dried to produce 224 g of freeze-driedpowders of Total salvianolic acid. The yield of the final product is4.5% based on the amount of the crude herb. The analysis according tothe method of Chinese Patent Application No. 01142288.2 filed onSeptember 2001 shows that, the totalphenolic acid amounts to 84.02%, andSalvianolic acid B is 54.17% in the final product.

EXAMPLE 4

Formula of Total Salvianolic Acid Capsule Total salvianolic acid 240 gMicrocrystalline cellulose  40 g Talcum powder 1.4% 3% ethanol solutionof polyvidone appropriate 1000 capsules are produced.

Total salvianolic acid and microcrystalline cellulose are mixedthoroughly. 3% ethanol solution of polyvidone is added into the mixtureto make soft stuff. It is sifted through 18-mesh screen sieve to givegranules, and dried at 60° C. for 30 to 45 min. Then, talcum powder isadded, and the mixture is stirred and filled into No. 1 capsule shell toproduce capsules each of which contains 240 mg.

EXAMPLE 5

Formula of of Total Salvianolic Acid Tablet Total salvianolic acid 240 gMicrocrystalline cellulose  40 g Talcum powder 1.4% 3% ethanol solutionof polyvidone appropriate 1000 tablets are produced.

Total salvianolic acid and microcrystalline cellulose are mixedthoroughly. 3% ethanol solution of polyvidone is added into the mixtureto make soft stuff. It is sifted through 18-mesh screen sieve to givegranules, and dried at 60° C. for 30 to 45 min. Then, talcum powders areadded, and the mixture is stirred and tableted.

EXAMPLE 6

Formula of Total Salvianolic Acid Granula Total salvianolic acid 240 gMicrocrystalline cellulose 40 g Talcum powder 1.4% 3% ethanol solutionof polyvidone appropriate 500 sachets are produced.

Total salvianolic acid and microcrystalline cellulose are mixedthoroughly. 3% ethanol solution of polyvidone is added into the mixtureto make soft stuff. It is sifted through 18-mesh screen sieve to givegranules, and dried at 60° C. for 30 to 45 min, and filled into sachets.

EXAMPLE 7

Formula of Total Salvianolic Acid Powder for Injection Total salvianolicacid 100 g Mannite  30 g Antallin  5 g Distilled water  5 ml

The above ingredients are mixed well, lyophilized, and filled into 1000vials.

1. A method for the preparation of total salvianolic acid comprising thesteps of: a. extracting Danshen with water; b. applying said Danshenextract on a polyamide column for separation; c. applying fractionsobtained above on a macroporous adsorption resin column for furtherseparation.
 2. The method as recited in claim 1, wherein said step (a)also comprises acidifying the extract and filtrating; said step (b)comprises applying said filtrate on a polyamide column, washing thecolumn with water to neutral condition, discarding the washings, elutingthe polyamide column with weak basic aqueous solution, and collectingthe fractions; said step (c) also comprises applying the basic fractionsof step (b) on macroporous adsorption resin column, after acidifying thefractions, firstly washing the column with water to neutral condition,discarding the washings, and then eluting the column with hydrous oranhydrous lower alcohol; the eluent being collected, concentrated underreduced pressure until no alcohol, and dried.
 3. The method as recitedin claim 2, wherein the heating extraction in step (a) by water isrepeated for 2-4 times, and for 0.5-2 h each.
 4. The method as recitedin claim 2, wherein the temperature for the said extraction of step (a)by water is 60-100° C.
 5. The method as recited in claim 2, wherein thetemperature for the said extraction of step (a) by water is 90-100° C.6. The method as recited in claim 2, wherein the said extract of step(a) is adjusted with an acid to a pH value of below 4, and the basicfractions of step (c) is acidified with an acid to a pH value of below4.
 7. The method as recited in claim 2, wherein the said extract of step(a) is acidified with an acid to a pH value of below 2, and the basicfractions of step (c) is acidified with hydrochloric acid or sulfuricacid to a pH value of below
 2. 8. The method as recited in claim 2,wherein in step (b) 0.01-2% of aqueous weak basic solution is used aseluant.
 9. The method as recited in claim 2, wherein in step (b)0.08-0.5% of aqueous solution of sodium hydrogen carbonate is used aseluant.
 10. The method as recited in claim 2, wherein the saidmacroporous adsorption resin of the step (c) is styrene type.
 11. Themethod as recited in claim 2, wherein the hydrous or anhydrous lowerC₁-C₅ alcohol is used as the eluant in the step (c).
 12. The method asrecited in claim 2, wherein in the step (c) the column is eluted withaqueous ethanol with a concentration of 40-95%.
 13. The method asrecited in claim 11, wherein the concentration of the lower C₁-C₅alcohol is 60-95%.
 14. The method as recited in claim 12, wherein theconcentration of the aqueous ethanol is 95%.
 15. A total salvianolicacid produced by the method of claim
 1. 16. a total salvianolic acid,produced by the following method: the Danshen herb is ground into crudepowder, and is extracted at 100° C. with water at the state of lightlyboiling for three times; wherein the herb is extracted for 1 hour withwater (×5.5 fold) added the first time, and extracted for 0.5 hour withwater (×3 fold) added the second and third times respectively; theextract is combined, and is acidified with 10% hydrochloric acid to a pHof 2.0; the extract is filtered, and the filtrate is loaded on polyamidecolumn; the column is washed with water (×5 fold) to neutral condition,and the washings are discarded; then the column is eluted with the 0.1%aqueous solution of sodium bicarbonate; the fractions are collected,acidified with 10% hydrochloric acid to pH 2.0, and is loaded on D101macro-porous adsorption resin column; the column is washed with water toneutral condition, and the washings are discarded; then the column iseluted with 95% ethanol, and the colored belt is collected when it iseluted down; the fractions are concentrated under the reduced pressureto entire dryness; the obtained product is dissolved with water; themixture is allowed to stand over night in refrigerator, and filtered bymicroporous membrane to give an extraction of Total salvianolic acid;immediately after this totalphenolic acid is adjusted with 2% sodiumhydroxide to pH 6.0, it is freeze dried to produce freeze-dried powdersof Total salvianolic acid.
 17. The total salvianolic acid according toclaim 15, wherein the content of the total salvianolic acid is more thanor equal to 80%, and the content of salvianolic acid B is more than orequal to 50%.
 18. A pharmaceutical composition, comprising the totalsalvianolic acid according to claim 1 as active ingredients, and apharmaceutically acceptable carrier or an excipient.
 19. A method forthe treatment of cardiovascular and cerebrovascular disease, nephrosis,hepatopathy, pneumonia, pneumocardial disease, pancreatitis, diabetesmellitus, cervical syndrome, ocular fundus vascular disease, ocularfundus neuro-disease, migrain, chronic gastritis, dizziness, orthopedicsdisease, mountain sickness and senile dementia comprising administratingpreparation of claim 15 to a patient.
 20. The method as recited in claim12, wherein the concentration of the aqueous ethanol is 60-95%.
 21. Thetotal salvianolic acid according to claim 16, wherein the content of thetotal salvianolic acid is more than or equal to 80%, and the content ofsalvianolic acid B is more than or equal to 50%.
 22. A method for thetreatment of cardiovascular and cerebrovascular disease, nephrosis,hepatopathy, pneumonia, pneumocardial disease, pancreatitis, diabetesmellitus, cervical syndrome, ocular fundus vascular disease, ocularfundus neuro-disease, migrain, chronic gastritis, dizziness, orthopedicsdisease, mountain sickness and senile dementia comprising administratingpreparation of claim 16 to a patient.